Magor- Topic 6: Exit of viruses Flashcards
Attachment step of poliovirus
That attachment step is part of the opening of the capsid, making a pore & releasing that RNA into the cytoplasm.
Assembly of poliovirus
- Virus attaches to pvr
- Uncoating at cell membrane
- Proteins are made as polyproteins
- Polyproteins are cleaved into their parts & synthesized
- Initial cleavage is autocatalyzed, but then proteases are released.
- To amplify this, that RNA is taken over to the outside of that vacuole
- (+) strands are replicated into (-) strands with RNA-dependent RNA polymerase
- The (-) strand is used to make more (+) strand
When does the assembly process of poliovirus begin?
Once there’s lots & lots of capsid proteins as well as lots of (+) strand RNAs around.
In poliovirus assembly, proteins are folded in the ________, then cleaved by _________
polyprotein, proteases
Intramolecular interactions are favored in the intact ________
polyprotein
RNA strand inside the capsid is doing the cleavage of ______
VP4
That’s brilliant because VP4 is inside the capsid and seals it up and makes it really solid. That step doesn’t happen unless there’s an RNA inside that capsid.
It’s the RNA itself that’s performing the catalysis.
Cleavage of VP0 to VP2 and VP4 requires the _______
RNA
How does the virus make sure that an RNA genome gets in every virion?
If the RNA is not in there, the final VP4 cleavage step doesn’t happen & the capsid just falls apart.
Empty capsids just sort of disassemble, reassemble, disassemble, reassemble all the time.
One of the polio vaccines that is in progress
One of the vaccines that is in progress is to make just the capsid proteins & trick it to stabilize the capsid proteins basically by recombinant DNA engineering.
They’re trying to make a vaccine in a way that doesn’t need any RNA. Get that capsid stable with just the capsid & then there will be no danger of it reverting to a live infectious virus.
Integrase and reverse transcriptase are packaged in virion.
Why does it do this?
So that it doesn’t have to make those proteins before it can make copies of itself & get integrated into the genome.
What happens once HIV is integrated into the genome?
once HIV is integrated into the genome, it can use the host transcriptional machinery to make copies of itself as well as it can use things like we do, like alternate splicing. That allows it to pack more in that genome.
gag gene
Transcription normally starts near the gag gene.
It transcribes through the gag gene, which encodes a few different proteins involved in the capsid.
And it will make a lot of those proteins because it needs a lot more capsid proteins than it does enzyme reverse transcription & integrase.
HIV integrates into genome then uses ______________ to make more copies.
host transcriptional machinery
For every 10X that HIV makes a transcript through gag, transcribing it with RNA polymerase, for the 10th time, what happens?
It will go through the Pol gene & make integrase & reverse transcriptase because it needs 10 capsid proteins for one of these enzymes.
Assembly of retroviral particles from a polyprotein precursor
These proteins are cleaved up, but they’re already in the order that they need to be in order to pile up & pack up and make these little units (MA=matrix, CA=capsid, NC=nucleocapsid) that will then go up to the membrane & get incorporated into that virion.
Where do the membrane proteins gp120 and gp41 get made?
- synthesized together as one protein & cleaved later
- proteins that were made inside the rough ER as integral membrane proteins, ultimately end up as surface proteins. And it’s the region that has all the surface proteins that’s where the viral assembly takes place.
Secreted or TM proteins are made on the ___________ and secreted into __________, or left in ER membrane
rough ER, ER lumen
Localization of viral proteins to the cellular membrane
- Rough ER buds off into Golgi, and Golgi buds off into rough vesicles that fuse with the cell membrane.
- Assembly of enveloped viruses frequently takes place at plasma membrane
When it fuses with the cell membrane, what was on the inside of that vesicle moving towards the membrane is now on the outside of the membrane.
DNA virus can be quite large
The larger the virus = the more _______________
the larger the virus= the more likely it is actually gonna bring in its own polymerase and its own RNA polymerase.
Smaller virus like parvovirus (ssDNA virus) uses ____________ and __________________
host DNA polymerase and host RNA polymerase
It isn’t using any of its genome to bring in a polymerase
Larger DNA viruses encode their own ________
enzymes
________ is the biggest DNA virus (~130-300 kb)
Pox
It brings in its own DNA polymerase & RNA polymerase
Why does the Pox virus bring in its own DNA polymerase & RNA polymerase?
They’re not as high fidelity as our polymerase and so that they can evolve.
That’s why it is advantageous for the virus to be big enough to bring in its own polymerase.
Herpes virus will bring in its own ___________ but it uses our __________________
DNA polymerase; host RNA polymerase
Structure and genome organization of Herpesvirus
-enveloped virus
- it has a long genome with about 200 genes. Some of those genes are involved in its lifecycle.
DNA viruses have to do a couple of things.
They’re the same orientation, the same kind of DNA as ours but our cells are not normally synthesizing DNA. We only do that in the S phase of our cell cycle. And so the virus first has to trick the cell into entering that S cycle.
What is the reason for that?
The reason for that is because the DNA polymerase is not necessarily around unless it is in that S cycle & the nucleotides (dNTPS, ATPs, etc.) those aren’t freely around unless the cell is in that synthesis phase.
Common mechanisms in exponential viral DNA replication
- Synthesis is template directed -you have to have a strand of DNA to make a strand of DNA
- Each strand is copied beginning at the origins
(replication origins= DNA replication regions; opens a bubble in the DNA) - Uses a DNA dependent DNA polymerase
Generation of dNTP for DNA synthesis is only present in ________
S phase
What is the most common pattern of replication of DNA of host and viral genomes
Bidirectional replication
Semidiscontinuous DNA synthesis from a bidirectional origin
- RNA synthesis is initiated on the strand by specialized enzyme called primase
- Synthesis of leading strand starts from replication origin on RNA primer
- Lagging strand proceeds in short pieces as template becomes open (Okazaki fragments)
We can’t initiate DNA synthesis without something for that incoming base to attack. It needs a __________ in order to put that base in place.
hydroxyl (OH) group
In the same way that the poliovirus use the tyrosine, what we do and what this virus does is
It synthesizes a little RNA using RNA primase
Discontinuous synthesis leaves _________ strand unfinished.
lagging
Removal of the primer form the 5’ end creates a gap that cannot be filled.
Successive rounds of replication result in the loss of genetic material, unless specialized mechanisms are in place to deal with it.
“End problem”
- If you make a little RNA primer & then begin your DNA synthesis, & then cleave out that RNA again, then you shorten that strand.
- We get away with that because we make big long chromosomes that are full of repetitive DNA. And so every time our cells replicate, that telomere does get a little shorter.
- Viruses could get really short very quickly if they didn’t have a way to solve this end problem. And every virus doe sit differently & one common mechanism is the rolling circle mechanism.
Rolling circle mechanism of DNA replication
- Viruses’ way of solving the end problem
- The virus circularizes its linear template
- An enzyme nicks that ds circle, revealing an OH group
- Replicate by continuous copying
- Displace strand
- Complete the lagging strand
- Double strand DNA templates result
- Keep making more copies by continuing around again
How do you get one genome in each capsid?
- Proteins bind to the ends of a genome unit
- Empty capsid binds to these proteins
- The DNA is stuffed in the capsid until it reaches those proteins at the other end
- Cleavage of DNA
Viral DNA synthesis occurs in specialized ___________
compartments
DNA templates and enzymes are sequestered into discrete sites in the nucleus.
Products of one replication cycle become templates for the next.
Host DNA polymerases proofread during DNA synthesis.
What does this mean?
It make a mistake= it chews it back with exonuclease activity and then goes forward correcting mistakes.
Viral DNA polymerases may not proofread to the same fidelity as ours. What is an advantage of this?
They can evolve
Acyclovir
- most of us are infected with Herpes simplex I which causes cold sores. Some people suffer from much worse cold sores than others. There is no vaccine, there is definitely no cure.
- There are drugs that can be used to reduce the symptoms.
- Acyclovir can be used to treat Herpes simplex symptoms and it is an inhibitor of the polymerase, preventing the DNA amplification steps.
Herpes simplex virus latent infection
In the latent infection, there are transcripts that are specifically used to keep that virus latent.
Many of those drugs then are reactivated by stress.
